We propose to examine the chemical nature of chromosomes which results in C-and G-banding. We have determined the reactions involved in DNA depolymerization and extraction during C-banding and used this chemical knowledge to devise a better C-banding method. By using chromosomes with DNA alternately labeled in eu-and heterochromatin, we will determine the rate limiting reaction responsible for retarding DNA extraction from C-bands. During G-banding, protein configurational changes in the fixed chromosome reduce the accessibility of DNA to Giemsa stain especially in interbands. Preliminary experiments indicate these changes are stabilized by ionic bonds. The proposed studies will examine the protein-DNA configurational changes occurring during G-banding. Large quantities of fixed chromosomes have been isolated and will be (1) fixed, (2) treated with trypsin or hot salt, (3) stained and (4) destained. The chemical composition and protein gel profiles of the chromosomes from each of these four stages will be determined as will protein removal, DNA accessibility, degree of protein denaturation and degree of ionic bonding to DNA. DNA accessibility will be measured with the probes methylene blue, alpha-dansyl-oligolysines of various DNA affinities and psoralin. Protein denaturation will be measured by availability of interior disulfides to reduction. Ionic binding of protein to DNA will be directly measured from the availability of epsilon-amino groups of lysine to react with dansyl chloride. The DNA from G-bands and R-bands has been isolated and will be characterized. These studies will examine a number of physical parameters of chromosomal DNA and protein which should permit a greater understanding of the structural significance of a visible chromosomal band. These studies have already and will continue to produce new banding techniques for the identification of congenital chromosomal defects.